Profiling Multiplexed Protein-Specific Sialylation in Cancer Drug Resistance Using Proximity Ligation Sequencing

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• Aberrant glycosylation, specifically increased sialylation of membrane proteins, is a key factor in cancer progression, influencing signaling, immune evasion, and treatment resistance.
• Current methods for studying protein-specific sialylation in living cells are limited in scope and scalability.

Purpose of the Study:

• To develop a novel, high-throughput method for analyzing protein-specific sialylation in living cells.
• To investigate the role of protein sialylation in cancer drug resistance.

Main Methods:

• SATP-seq (Sialic acid-reactive probe and Targeted Protein probe sequencing) was developed, integrating metabolic glycan labeling with DNA-programmed proximity ligation.
• This method uses nanobodies or aptamers for dual recognition of sialic acids and protein epitopes, converting glycosylation information into DNA barcodes for sequencing.
• Applied to gefitinib-sensitive (PC9) and resistant (PC9GR) lung cancer cells to profile seven membrane glycoproteins.

Main Results:

• SATP-seq successfully enabled multiplexed and quantitative profiling of protein-specific sialylation.
• Significant differences in sialylation patterns were observed between sensitive and resistant cancer cells.
• Differential sialylation of Epidermal Growth Factor Receptor (EGFR) and CD47 was identified as a potential mechanism in gefitinib resistance.

Conclusions:

• SATP-seq is a scalable and powerful tool for dissecting the complex role of protein sialylation in cancer drug resistance.
• Targeted analysis revealed specific sialylation changes in EGFR and CD47, highlighting their involvement in resistance pathways.
• This approach provides new insights into glycosylation-driven mechanisms underlying therapeutic failure in cancer.